Microtubules (MT) are tubular protein structures found in all eucaryotic cells. They are involved in cell motility cell transport, chromosome segregation during mitosis, and maintenance of normal cell structure. MT are composed primarily of two closely related protein subunits termed alpha and beta tubulin (Tu). The MT protomer is a heterologous dimer consisting of an alpha and beta Tu. In vivo, MT exist in a dynamic equilibrium with the Tu protomer. A number of agents have been found to alter MT structure, both in vivo and in vitro. These include low temperature, nucleotides, divalent cations, and plant alkaloids such as colchicine and vinblastine. Many of these agents bind to Tu and alter its in vitro assembly properties. Using a combination of biochemical, fluorescence, and rapid kinetic techniques, the interaction of Tu with nucleotides, divalent cations, and colchicine will be explored. Affinity labeling techniques will be used to determine which Tu subunit contains the rapidly exchangeable GTP binding site. Equilibrium parameters for binding of these ligands will be determined by fluorescence titration and equilibrium gel filtration. Fluorescence stopped-flow studies of the interaction of Tu with GTP, Ca, and colchicine will be performed to determine the kinetic parameters for interaction and the molecular mechanisms involved. Studies of chemical reactivity, dynamic fluorescence quenching, and dynamic fluorescence anisotropy will be performed to determine whether these ligands cause conformational changes in Tu and if so the nature of these changes. These studies will be correlated with other studies on the dynamics of Tu disassembly, both in the presence and absence of these ligands. Analysis of the data obtained should provide insight into the molecular mechanism whereby Tu assembly is regulated.